Trace detection and physicochemical characterization of protein aggregates have a large impact in understanding and diagnosing many diseases, such as ageing-related neurodegeneration and systemic amyloidosis, for which the formation of protein aggregates is one of the pathological hallmarks. Here we demonstrate an innovative label-free method for detecting and characterizing small amounts of early stage protein aggregates using a Raman active nanofluidic device. Sub-micrometre channels formed by a novel elastomeric collapse technique enable the separation and concentration of matured protein aggregates from small protein molecules. The Raman enhancement by gold nanoparticle clusters fixed below a micro/nanofluidic junction allows characterization of intrinsic properties of protein aggregates at concentration levels (∼fM) much lower than can be done with traditional analytical tools. With our device we show for the first time the concentration dependence of protein aggregation over these low concentration ranges. We expect that our method could facilitate definitive diagnosis and possible therapeutics of diseases at early stages.